The invention concerns a method for the interference-free determination of iron in biological samples, in particular in serum in which bound iron is released, the released iron is reduced to Fe.sup.2+, a color reagent solution is added and a color complex that forms is measured photometrically and it also concerns a combination of reagents which is suitable for the interference-free determination of iron especially in the presence of high amounts of EDTA.
Disorders of iron metabolism, in particular iron deficiency and disorders of iron absorption are widespread especially in the female population. Hence the detection of iron in body fluids and in particular in serum is one of the standard determinations in medical analytics. Iron is provided with the diet and absorbed via the intestinal mucosa. Bound to transferrin in a trivalent state, it is then transported to the bone marrow where it is mainly incorporated into haemoglobin. Anaemic symptoms occur if too little iron is absorbed.
The determination of iron in serum is one of the most frequently carried out trace element analyses in clinical diagnostics. With regard to the release of iron from transferrin, two basic test variants for the determination are known. In the one case iron can be detached by adding detergent mixtures (e.g. EP 0 130 537), guanidinium chloride (e.g. EP 0 343 592) or for example urea-containing denaturing agents (e.g. DE 44 01 754) at a weakly acidic pH. On the other hand, iron can be detached from transferrin in a strongly acidic medium, in particular at a pH value of ca. 2.5 or lower (Clin. Chem. vol. 26 (1980), 327-331).
However, a disadvantage of the said test principles is that the presence of EDTA which is often present in biological samples, for example as a result of corresponding pretreatments, causes interference. The reason is that EDTA is able to form complexes with iron and the iron bound in this manner is therefore not available for the formation of a color complex. This can lead to false-negative iron values especially with EDTA plasma as a sample material. Furthermore, there is a risk that when such determinations are carried out on automated analysers, there is a carry-over of EDTA from other test liquids due to unavoidable pipetting or stirring devices or inadequately cleaned reaction vessels which results in a reduced color signal and thus to a reduced recovery of the iron.
In order to eliminate this disadvantage, zinc (II) salts are added in test variants which release the iron by a suitable denaturing agent. The EDTA contamination is masked by Zn.sup.2+ ions which avoids interference due to reactions of EDTA with Fe.sup.2+ ions.
In contrast the addition of zinc(II) salts does not eliminate interference caused by EDTA in test variants in which iron is released in a strongly acidic medium. Furthermore it has turned out that in the presence of high salt concentrations (ca. 4 mol/l) and/or high detergent concentrations (ca. 7%) the reagent solution is observed to "creep out" of the reagent bottles leading for example to destruction of bar code labels and corrosion of metal components in automated analysers.
Consequently the object of the present invention is to provide a method and reagent for the determination of iron in biological sample material which is suitable for the release of iron at strongly acidic pH values and/or in solutions with high salt or detergent concentrations in which no essential interference occurs in the presence of EDTA and reduced recovery of iron caused by EDTA is avoided.
The object is achieved by a method for the determination of iron in a biological sample in which the bound iron is released, the released iron is reduced to Fe.sup.2+, a color reagent solution is added and the color complex that is formed is measured photometrically, wherein the sample is contacted with a water-soluble salt composed of metal cations of the IIIrd main group and subgroup (III a+b) of the periodic system of the elements such as scandium, indium, lanthanum or gallium. Indium and/or scandium salts are preferred according to the invention. The counterion of the salts to be added according to the invention is uncritical provided water-soluble salts are formed. In particular divalent or trivalent cations and the following acidic residues have proven to be suitable: halogenides, phosphates, nitrates, sulfites and sulfates, of which chlorides and sulfates are preferred.
The concentration of the salts according to the invention of the IIIrd main group or subgroup in the reagent solution (final concentration in the test) is at least about 0.1 mmol/l, a range between about 0.1 and 50 mmol/l is preferred and a concentration between about 1 and 20 mmol/l is particularly preferred.